Cellular behavior is commonly controlled by signals in the extracellular environment. The effects can be dramatic, such as the decision to proliferate or to differentiate, and so the responses must be highly regulated and carefully orchestrated. While many signaling pathways and their molecular components have been identified, some well-studied systems offer a unique opportunity to test how multiple components achieve a unified cellular response that integrates information from different subcellular regions as well as information about the physiological status of the cell. The signal transduction pathway that is activated by extracellular mating pheromones in the yeast Saccharomycescerevisiae presents an ideal model system in which to probe the intricacies that are built into a cellular response. Signaling in this system involves the dynamic assembly of plasma membrane-localized signaling complexes, which include proteins found ubiquitously in a variety of signaling pathwaysfrom yeast to humans, such as a PAK-family kinase, a heterotrimeric G protein, a MAP kinase cascade, and a scaffold protein. Here we make use of a rich foundation of information and genetic reagents to test several intriguing models about the mechanisms that propagate intracellular signaling and their interface with cell biological structures, with particular emphasis on the role of subcellular localization. One goal will be to determine how membrane recruitment of the MAP kinase cascade scaffold protein Ste5 amplifies signaling through the kinase cascade, and whether this signaling occurs predominantly between or within individual scaffold molecules. Another goal will be to dissect the multiple routes by which the mating MAP kinase cascade can inhibit the mitotic cell cycle, and to determine why it is important for this signaling pathway to be inactivated as cells commit to division. Also under investigation will be the role of a small membrane-binding domain in the PAK-family kinase Ste20, and the prevalence of similar domains in other polarized proteins. Overall, we expect these studies to contribute to a sophisticated understanding of signal transduction by revealing mechanisms that are built into signaling systems to allow dynamic and integrative responses within a cell.